Cilostazol is an effective causal therapy for preventing paclitaxel-induced peripheral neuropathy by suppression of Schwann cell dedifferentiation.

Chemotherapy-induced peripheral neuropathy (CIPN) can lead to discontinuation of chemotherapy and is consequently a serious impediment to effective cancer treatment. Due to our limited understanding of mechanisms underlying the pathogenesis of CIPN, no causal therapy has been approved for relief of this condition. We previously demonstrated that taxanes (paclitaxel and docetaxel) induce Schwann cell dedifferentiation, characterized by increased expression of p75 and galectin-3, ultimately leading to demyelination. These changes appear to be responsible for CIPN pathogenesis. This study was designed to identify a novel candidate therapeutic for CIPN with the ability to suppress paclitaxel-induced Schwann cell dedifferentiation. Given that elevation of cyclic adenosine monophosphate (cAMP) signaling participates in Schwann cell differentiation, we performed immunocytochemical screening of phosphodiesterase (PDE) inhibitors. We found that the PDE3 inhibitor cilostazol strongly promoted differentiation of primary cultures of rat Schwann cells via a mechanism involving cAMP/exchange protein directly activated by cAMP (Epac) signaling. Co-treatment with cilostazol prevented paclitaxel-induced dedifferentiation of Schwann cell cultures and demyelination in a mixed culture of Schwann cells and dorsal root ganglia neurons. Notably, continuous oral administration of cilostazol suppressed Schwann cell dedifferentiation within the sciatic nerve and the development of mechanical hypersensitivity in a mouse model of paclitaxel-related CIPN. Importantly, cilostazol potentiated, rather than inhibited, the anti-cancer effect of paclitaxel on the human breast cancer cell line MDA-MB-231. These findings highlight the potential utility of cilostazol as a causal therapeutic that avoids the development of paclitaxel-related CIPN without compromising anti-cancer properties.

Pronociceptive Roles of Schwann Cell-Derived Galectin-3 in Taxane-Induced Peripheral Neuropathy.

Chemotherapy-induced peripheral neuropathy (CIPN) is a severe dose-limiting side effect of taxanes such as paclitaxel and docetaxel. Despite the high medical needs, insufficient understanding of the complex mechanism underlying CIPN pathogenesis precludes any endorsed causal therapy to prevent or relieve CIPN. In this study, we report that elevation of plasma galectin-3 level is a pathologic change common to both patients with taxane-treated breast cancer with CIPN and a mouse model of taxane-related CIPN. Following multiple intraperitoneal injections of paclitaxel in mice, galectin-3 levels were elevated in Schwann cells within the sciatic nerve but not in other peripheral organs or cells expressing galectin-3. Consistent with this, paclitaxel treatment of primary cultures of rat Schwann cells induced upregulation and secretion of galectin-3. In vitro migration assays revealed that recombinant galectin-3 induced a chemotactic response of the murine macrophage cell line RAW 264.7. In addition, perineural administration of galectin-3 to the sciatic nerve of naive mice mimicked paclitaxel-induced macrophage infiltration and mechanical hypersensitivity. By contrast, chemical depletion of macrophages by clodronate liposomes suppressed paclitaxel-induced mechanical hypersensitivity despite the higher level of plasma galectin-3. Deficiency (Galectin-3 -/- mice) or pharmacologic inhibition of galectin-3 inhibited paclitaxel-induced macrophage infiltration and mechanical hypersensitivity. In conclusion, we propose that Schwann cell-derived galectin-3 plays a pronociceptive role via macrophage infiltration in the pathogenesis of taxane-induced peripheral neuropathy. Therapies targeting this phenomenon, which is common to patients with CIPN and mouse models, represent a novel approach to suppress taxane-related CIPN. SIGNIFICANCE: These findings demonstrate that the elevation of plasma galectin-3 is a CIPN-related pathologic change common to humans and mice, and that targeting galectin-3 is a therapeutic option to delay CIPN progression.

T-cell responses to sequentially emerging viral escape mutants shape long-term HIV-1 population dynamics.

HIV-1 strains harboring immune escape mutations can persist in circulation, but the impact of selection by multiple HLA alleles on population HIV-1 dynamics remains unclear. In Japan, HIV-1 Reverse Transcriptase codon 135 (RT135) is under strong immune pressure by HLA-B*51:01-restricted and HLA-B*52:01-restricted T cells that target a key epitope in this region (TI8; spanning RT codons 128-135). Major population-level shifts have occurred at HIV-1 RT135 during the Japanese epidemic, which first affected hemophiliacs (via imported contaminated blood products) and subsequently non-hemophiliacs (via domestic transmission). Specifically, threonine accumulated at RT135 (RT135T) in hemophiliac and non-hemophiliac HLA-B*51:01+ individuals diagnosed before 1997, but since then RT135T has markedly declined while RT135L has increased among non-hemophiliac individuals. We demonstrated that RT135V selection by HLA-B*52:01-restricted TI8-specific T-cells led to the creation of a new HLA-C*12:02-restricted epitope TN9-8V. We further showed that TN9-8V-specific HLA-C*12:02-restricted T cells selected RT135L while TN9-8T-specific HLA-C*12:02-restricted T cells suppressed replication of the RT135T variant. Thus, population-level accumulation of the RT135L mutation over time in Japan can be explained by initial targeting of the TI8 epitope by HLA-B*52:01-restricted T-cells, followed by targeting of the resulting escape mutant by HLA-C*12:02-restricted T-cells. We further demonstrate that this phenomenon is particular to Japan, where the HLA-B*52:01-C*12:02 haplotype is common: RT135L did not accumulate over a 15-year longitudinal analysis of HIV sequences in British Columbia, Canada, where this haplotype is rare. Together, our observations reveal that T-cell responses to sequentially emerging viral escape mutants can shape long-term HIV-1 population dynamics in a host population-specific manner.

Schwann cell-derived CXCL1 contributes to human immunodeficiency virus type 1 gp120-induced neuropathic pain by modulating macrophage infiltration in mice.

The neuroinflammatory responses to human immunodeficiency virus type 1 (HIV-1) coat proteins, such as glycoprotein 120 (gp120), are considered to be responsible for the HIV-associated distal sensory neuropathy. Accumulating evidences suggest that T-cell line tropic X4 gp120 increases macrophage infiltration into the peripheral nerves, and thereby induces neuroinflammation leading to pain. However, the mechanisms underlying X4 gp120-induced macrophage recruitment to the peripheral nervous systems remain unclear. Here, we demonstrated that perineural application of X4 gp120 from HIV-1 strains IIIB and MN elicited mechanical hypersensitivity and spontaneous pain-like behaviors in mice. Furthermore, flow cytometry and immunohistochemical studies revealed increased infiltration of bone marrow-derived macrophages into the parenchyma of sciatic nerves and dorsal root ganglia (DRG) 7 days after gp120 IIIB or MN application. Chemical deletion of circulating macrophages using clodronate liposomes markedly suppressed gp120 IIIB-induced pain-like behaviors. In in vitro cell infiltration analysis, RAW 264.7 cell (a murine macrophage cell line) was chemoattracted to conditioned medium from gp120 IIIB- or MN-treated cultured Schwann cells, but not to conditioned medium from these gp120-treated DRG neurons, suggesting possible involvement of Schwann cell-derived soluble factors in macrophage infiltration. We identified using a gene expression array that CXCL1, a chemoattractant of macrophages and neutrophils, was increased in gp120 IIIB-treated cultured Schwann cells. Similar to gp120 IIIB or MN, perineural application of recombinant CXCL1 elicited pain-like behaviors accompanied by macrophage infiltration to the peripheral nerves. Furthermore, the repeated injection of CXCR2 (receptor for CXCL1) antagonist or CXCL1 neutralizing antibody prevented both pain-like behaviors and macrophage infiltration in gp120 IIIB-treated mice. Thus, the present study newly defines that Schwann cell-derived CXCL1, secreted in response to X4 gp120 exposure, is responsible for macrophage infiltration into peripheral nerves, and is thereby associated with pain-like behaviors in mice. We propose herein that communication between Schwann cells and macrophages may play a prominent role in the induction of X4 HIV-1-associated pain.

Improvement of peripheral vascular impairment by a phosphodiesterase type 5 inhibitor tadalafil prevents oxaliplatin-induced peripheral neuropathy in mice.

Oxaliplatin, a platinum-based chemotherapeutic drug, frequently induces peripheral neuropathy. Accumulating evidences suggest a possible relationship between peripheral vascular impairment and peripheral neuropathy. In this study, we investigated the effects of vasodilators on cumulative peripheral neuropathy induced by repeated injections of oxaliplatin (10 mg/kg) once a week for 8 weeks in mice. Single injections of vasodilators, including a phosphodiesterase type 5 inhibitor tadalafil acutely alleviated oxaliplatin-induced cold hypersensitivity, while tadalafil had no effect on the mechanical hypersensitivity. By contrast, long-term administration of tadalafil (0.1% in chow diets) during the oxaliplatin injection period reduced the oxaliplatin-induced decreases in skin temperature and blood flow without affecting platinum concentrations in blood, sciatic nerves, and dorsal root ganglion. The long-term administration significantly suppressed cold, mechanical, and electrical current hypersensitivities as well as thermal hypoesthesia. Furthermore, it prevented the decreases in sensory nerve conductance velocity and the number of endoneurial microvessels, and axon degeneration in the sciatic nerves. In vitro studies confirmed that tadalafil does not interfere with the cytotoxicity of oxaliplatin against human cancer cell lines. Altogether, these results suggest that improvement of peripheral vascular impairment by tadalafil could alleviate and prevent oxaliplatin-induced peripheral neuropathy.

[Identification of biomarkers and new therapies for taxane-induced peripheral neuropathy].

Chemotherapy-induced peripheral neuropathy (CIPN) is a side effect frequently caused by taxanes. Because the mechanisms underlying CIPN pathogenesis remain to be fully elucidated, there is no indicator for objective diagnosis like a biomarker. In addition, treatment options for CIPN is still unsatisfactory. We have previously demonstrated that paclitaxel preferentially impair myelin-forming Schwann cells, and consequently induce dedifferentiation and demyelination of Schwann cells. Recently, in a paclitaxel CIPN model mouse, we found that an inflammatory factor is released from dedifferentiated Schwann cells in the mouse sciatic nerve into the blood, highly correlated with the on-set of mechanical hypersensitivity. On the other hand, considering our previous findings, it seems that some drugs, which supply newly formed mature Schwann cells at the sites of demyelinated lesions, may be a new beneficial therapy for taxane-induced peripheral neuropathy. In this review, we will introduce our findings about new therapeutic drug candidate for taxane-related CIPN based on this concept, and plasma biomarker to detect CIPN on-set and progression.

Comparative Analysis of Bone Marrow-derived Mast Cell Differentiation in C57BL/6 and BALB/c Mice.

Allergic diseases have increased in the last three decades. Mast cells play a critical role in allergic diseases along with allergen-specific immunoglobulin E (IgE). Following mast cell degranulation elicited by ligation of the IgE-FcεRI receptor complex with allergen, allergic reactions are followed by various symptoms such as vascular hyperpermeability, mucous secretion, itching, sneezing, wheezing, rashes, fever, and anaphylactic shock. Susceptibility or inclination to allergy varies depending on individual genetic traits and living environment, and it has long been believed that such an inclination is determined by an immunologic balance of T helper cell types. Mouse strains also have different susceptibilities to allergy. Similar to T helper cells and macrophages, it is not known whether mast cells can also be divided into two different types between mouse strains. In this study, we prepared bone marrow-derived mast cells from BALB/c and C57BL/6 mice and examined their cellular properties. Cellular response to IL-3 and the process of mast cell differentiation from bone marrow cells were different on the basis of cell surface marker molecules. BALB/c-derived cells more efficiently exhibited degranulation than did C57BL/6-derived cells following both calcium ionophore and receptor crosslinking. These functional differences persisted even after a longer cell culture for 8 weeks, suggesting a difference in cell-autonomous characteristics. These results support the concept that mast cells also have different cell types dependent on their genetic background. Abbreviations: Ab: antibody; BMMC: bone marrow-derived mast cell; DNP: dinitrophenyl; FACS: fluorescence-activated cell sorter; FCS: fetal calf serum; FITC: fluorescein isothiocyanate; FSC: forward scatter; HRP: horseradish peroxidase; HSA: human serum albumin; Ig: immunoglobulin; IL: interleukin; MIP-2: macrophage inflammatory protein-2; MCP: mast cell protease; PE: phycoerythrin; PerCP: Peridinin chlorophyll protein complex; SNP: single nucleotide polymorphisms; SSC: side scatter; Th: T helper; TNF-α: tumor necrosis factor alpha.

A SNP uncoupling Mina expression from the TGFß signaling pathway.

INTRODUCTION: Mina is a JmjC family 2-oxoglutarate oxygenase with pleiotropic roles in cell proliferation, cancer, T cell differentiation, pulmonary inflammation, and intestinal parasite expulsion. Although Mina expression varies according to cell-type, developmental stage and activation state, its transcriptional regulation is poorly understood. Across inbred mouse strains, Mina protein level exhibits a bimodal distribution, correlating with inheritance of a biallelic haplotype block comprising 21 promoter/intron 1-region SNPs. We previously showed that heritable differences in Mina protein level are transcriptionally regulated. METHODS: Accordingly, we decided to test the hypothesis that at least one of the promoter/intron 1-region SNPs perturbs a Mina cis-regulatory element (CRE). Here, we have comprehensively scanned for CREs across a Mina locus-spanning 26-kilobase genomic interval. RESULTS: We discovered 8 potential CREs and functionally validated 4 of these, the strongest of which (E2), residing in intron 1, contained a SNP whose BALB/c-but not C57Bl/6 allele-abolished both Smad3 binding and transforming growth factor beta (TGFß) responsiveness. CONCLUSIONS: Our results demonstrate the TGFß signaling pathway plays a critical role in regulating Mina expression and SNP rs4191790 controls heritable variation in Mina expression level, raising important questions regarding the evolution of an allele that uncouples Mina expression from the TGFß signaling pathway.

Taxanes and platinum derivatives impair Schwann cells via distinct mechanisms.

Impairment of peripheral neurons by anti-cancer agents, including taxanes and platinum derivatives, has been considered to be a major cause of chemotherapy-induced peripheral neuropathy (CIPN), however, the precise underlying mechanisms are not fully understood. Here, we examined the direct effects of anti-cancer agents on Schwann cells. Exposure of primary cultured rat Schwann cells to paclitaxel (0.01 µM), cisplatin (1 µM), or oxaliplatin (3 µM) for 48 h induced cytotoxicity and reduced myelin basic protein expression at concentrations lower than those required to induce neurotoxicity in cultured rat dorsal root ganglion (DRG) neurons. Similarly, these anti-cancer drugs disrupted myelin formation in Schwann cell/DRG neuron co-cultures without affecting nerve axons. Cisplatin and oxaliplatin, but not paclitaxel, caused mitochondrial dysfunction in cultured Schwann cells. By contrast, paclitaxel led to dedifferentiation of Schwann cells into an immature state, characterized by increased expression of p75 and galectin-3. Consistent with in vitro findings, repeated injection of paclitaxel increased expression of p75 and galectin-3 in Schwann cells within the mouse sciatic nerve. These results suggest that taxanes and platinum derivatives impair Schwan cells by inducing dedifferentiation and mitochondrial dysfunction, respectively, which may be important in the development of CIPN in conjunction with their direct impairment in peripheral neurons.

Effects of protein tyrosine phosphatase-PEST are reversed by Akt in T cells.

T cell activation is regulated by a balance between phosphorylation and dephosphorylation that is under the control of kinases and phosphatases. Here, we examined the role of a non-receptor-type protein tyrosine phosphatase, PTP-PEST, using retrovirus-mediated gene transduction into murine T cells. Based on observations of vector markers (GFP or Thy1.1), exogenous PTP-PEST-positive CD4(+) T cells appeared within 2 days after gene transduction; the percentage of PTP-PEST-positive cells tended to decrease during a resting period in the presence of IL-2 over the next 2 days. These vector markers also showed much lower expression intensities, compared with control cells, suggesting a correlation between the percent reduction and the low marker expression intensity. A catalytically inactive PTP-PEST mutant also showed the same tendency, and stepwise deletion mutants gradually lost their ability to induce the above phenomenon. On the other hand, these PTP-PEST-transduced cells did not have an apoptotic phenotype. No difference in the total cell numbers was found in the wells of a culture plate containing VEC- and PTP-PEST-transduced T cells. Moreover, serine/threonine kinase Akt, but not the anti-apoptotic molecules Bcl-2 and Bcl-XL, reversed the phenotype induced by PTP-PEST. We discuss the novel mechanism by which Akt interferes with PTP-PEST.

Transcriptional activation of Mina by Sp1/3 factors.

Mina is an epigenetic gene regulatory protein known to function in multiple physiological and pathological contexts, including pulmonary inflammation, cell proliferation, cancer and immunity. We showed previously that the level of Mina gene expression is subject to natural genetic variation linked to 21 SNPs occurring in the Mina 5' region. In order to explore the mechanisms regulating Mina gene expression, we set out to molecularly characterize the Mina promoter in the region encompassing these SNPs. We used three kinds of assays--reporter, gel shift and chromatin immunoprecipitation--to analyze a 2 kb genomic fragment spanning the upstream and intron 1 regions flanking exon 1. Here we discovered a pair of Mina promoters (P1 and P2) and a P1-specific enhancer element (E1). Pharmacologic inhibition and siRNA knockdown experiments suggested that Sp1/3 transcription factors trigger Mina expression through additive activity targeted to a cluster of four Sp1/3 binding sites forming the P1 promoter. These results set the stage for comprehensive analysis of Mina gene regulation from the context of tissue specificity, the impact of inherited genetic variation and the nature of upstream signaling pathways.

Differential clade-specific HLA-B*3501 association with HIV-1 disease outcome is linked to immunogenicity of a single Gag epitope.

The strongest genetic influence on immune control in HIV-1 infection is the HLA class I genotype. Rapid disease progression in B-clade infection has been linked to HLA-B*35 expression, in particular to the less common HLA-B*3502 and HLA-B*3503 subtypes but also to the most prevalent subtype, HLA-B*3501. In these studies we first demonstrated that whereas HLA-B*3501 is associated with a high viral set point in two further B-clade-infected cohorts, in Japan and Mexico, this association does not hold in two large C-clade-infected African cohorts. We tested the hypothesis that clade-specific differences in HLA associations with disease outcomes may be related to distinct targeting of critical CD8(+) T-cell epitopes. We observed that only one epitope was significantly targeted differentially, namely, the Gag-specific epitope NPPIPVGDIY (NY10, Gag positions 253 to 262) (P = 2 × 10(-5)). In common with two other HLA-B*3501-restricted epitopes, in Gag and Nef, that were not targeted differentially, a response toward NY10 was associated with a significantly lower viral set point. Nonimmunogenicity of NY10 in B-clade-infected subjects derives from the Gag-D260E polymorphism present in ∼90% of B-clade sequences, which critically reduces recognition of the Gag NY10 epitope. These data suggest that in spite of any inherent HLA-linked T-cell receptor repertoire differences that may exist, maximizing the breadth of the Gag-specific CD8(+) T-cell response, by the addition of even a single epitope, may be of overriding importance in achieving immune control of HIV infection. This distinction is of direct relevance to development of vaccines designed to optimize the anti-HIV CD8(+) T-cell response in all individuals, irrespective of HLA type.

Selection of HLA-B57-associated Gag A146P mutant by HLA-B∗48:01-restricted Gag140-147-specific CTLs in chronically HIV-1-infected Japanese.

We previously showed the possibility that Gag A146P, which is an escape mutant from HLA-B∗57-restricted CTLs, was selected by HLA-B∗48:01-restricted Gag138-147(LI10)-specific CTLs in a Japanese cohort in which HLA-B∗57 individuals were not detected. We herein demonstrated Gag140-147(GI8) to be the optimal epitope rather than LI10 and that GI8-specific T cells failed to recognize the A146P mutant virus-infected cells. The sequence analysis of Gag146 in 261 chronically HIV-1-infected Japanese showed the accumulation of the A146P mutation in HLA-B∗48:01(+) individuals. These findings together indicate that the A146P mutant is accumulating in Japanese by selection by GI8-specific CTLs.

Cutting edge: Dab2 is a FOXP3 target gene required for regulatory T cell function.

FOXP3-expressing regulatory T (Treg) cells are vital for maintaining peripheral T cell tolerance and homeostasis. The mechanisms by which FOXP3 target genes orchestrate context-dependent Treg cell function are largely unknown. In this study we show that in mouse peripheral lymphocytes the Drosophila Disabled-2 (Dab2) homolog, a gene that is involved in enhancing TGFbeta responses, is exclusively expressed in FOXP3+ regulatory T cells. Dab2 is a direct target of FOXP3, and regulatory T cells lacking DAB2 are functionally impaired in vitro and in vivo. However, not all aspects of Treg cell function are perturbed, and DAB2 appears to be dispensable for Treg cell function in maintaining naive T cell homeostasis.

Mina, an Il4 repressor, controls T helper type 2 bias.

T helper type 2 (T(H)2) bias, which is the propensity of naive CD4(+) T cells to differentiate into interleukin 4 (IL-4)-secreting T(H)2 cells, is a genetic trait that affects susceptibility to infectious, autoimmune and allergic diseases. T(H)2 bias correlates with the amount of IL-4 initially secreted by newly activated helper T cells that feeds back positively through the pathway of the IL-4 receptor and the transcription factors STAT6 and GATA-3 to drive T(H)2 development. Here we identify Mina, a member of the jumonji C (JmjC) protein family, as a genetic determinant of T(H)2 bias. Mina specifically bound to and repressed the Il4 promoter. Mina overexpression in transgenic mice impaired Il4 expression, whereas its knockdown in primary CD4(+) T cells led to Il4 derepression. Our findings collectively provide mechanistic insight into an Il4-regulatory pathway that controls helper T cell differentiation and genetic variation in T(H)2 bias.

Long-term exposure to superantigen induces p27Kip1 and Bcl-2 expression in effector memory CD4+ T cells.

The long-term exposure of mice to superantigen SEA using a mini-osmotic pump (SEA pump) induced a long-lasting expansion of Vbeta3+ CD4+ T cells with T helper (Th) 2 cell-type properties. Removal of the SEA pump 10 days after pump implantation did not significantly alter the level of Vbeta3+ CD4+ T cell expansion/maintenance. Furthermore, CFSE-labeled CD4+ T cells failed to divide when transferred to post-implantation day 15 mice. Thus, CD4+ T cells appeared to survive for at least 30 days in the absence of a sufficient amount of antigen to trigger cell division. STAT6 deficient mice, in which Th2 cell development is largely impaired, also exhibited a protracted cell expansion, similar to that observed in normal mice, suggesting that the Th2 cell property is dispensable for the maintenance of Vbeta3+ CD4+ T cell expansion. The expanded CD4+ T cells on post-implantation day 26 were arrested in the G0/G1 phase of the cell cycle and showed a lower level of cell division upon restimulation. The Cdk inhibitor p27(Kip1) was highly expressed, and Cdk2 was downregulated. Moreover, the CD4+ T cells were resistant to in vitro apoptosis induction in parallel with their level of Bcl-2 expression. Collectively, the Vbeta3+ CD4+ T cells appeared to develop into long-lived memory T cells with cell cycle arrest upon long-term exposure to SEA.